Large Domain Movements Upon UvrD Dimerization and Helicase Activation

Abstract

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.single molecule | conformational heterogeneity | DNA motors | DNA repair U vrD from Escherichia coli is a nonhexameric SF1A DNA helicase/translocase, structurally similar to E. coli Rep and Bacillus stearothermophilus PcrA, which is involved in many aspects of genome maintenance (1, 2), including DNA repair (3, 4), replication (5-8), and recombination (7, 9-11). Its ATPdependent activities include translocation along single-stranded (ss) DNA (12-16), duplex DNA unwinding (17-22), displacement of RecA filaments from ssDNA (10, 11), and pushing of proteins along ssDNA (23).The activities of UvrD can be modulated in vitro by its assembly state and/or by binding partners (1). UvrD monomers can translocate directionally (3′ to 5′) along ssDNA [∼190 nucleotides (nts) s −1 ] (12, 13, 15, 16), in a reaction that is tightly coupled to ATP hydrolysis (14). However, UvrD monomers show little helicase activity (12,16,21,24,25). In the absence of accessory proteins, helicase activity in vitro requires formation of a UvrD dimer (16,21,(25)(26)(27). The dimer unwinds dsDNA with rates of ∼70 base pair (bp) s −1 (20,21,24,27). Rep and PcrA monomers also show no helicase activity and require oligomerization or an accessory protein for helicase activity in vitro (28-31).UvrD, Rep, and PcrA all contain four subdomains (1A, 2A, 1B, and 2B) (2,(32)(33)(34)(35), and the 2B subdomain can undergo a substantial rotation about a hinge region connecting it to the 2A subdomain (2,22,(32)(33)(34)(35)(36)(37). A structure of an apo UvrD monome...

UvrD helicase activation by MutL involves rotation of its 2B subdomain

“…A crystal structure of a UvrD monomer complexed with a 3′-(dN) 7 partial duplex DNA (18 to 28 bp) shows the 2B subdomain in a closed state and in direct contact with duplex DNA (22). However, ensemble and singlemolecule FRET solution studies (23,24) show that UvrD monomers bound to a 3′-(dT) 20 -duplex DNA substrate of 18 bp display a distribution of 2B subdomain rotational conformational states that center on a more open state. We investigated the distribution of 2B subdomain states for UvrD bound to a partial ss-ds DNA by using single-molecule FRET.…”

“…We investigated the distribution of 2B subdomain states for UvrD bound to a partial ss-ds DNA by using single-molecule FRET. The rotational state of the 2B subdomain was probed by using a previously characterized double-cysteine UvrD mutant, UvrDΔCys-(A100C, A473C), referred to as UvrD-DM-1B/2B, with cysteines in the 1B (A100C) and 2B subdomains (A473C) (23,24) (Fig. 1A).…”

“…2 Cys residues were labeled stochastically with a mixture of Cy3 (donor) and Cy5 (acceptor) fluorophores as described previously (23,24). As predicted from the distances between residues A100 and A473 in the crystal structures of apo UvrD (23) and UvrD in complex with partial duplex DNA (22), and as shown previously in solution (23,24), the Cy3/Cy5-labeled UvrD-DM-1B/2B construct, hereafter referred to as Cy3/Cy5-UvrD, yields a high FRET efficiency signal, E FRET , when the 2B subdomain is in a closed state and a low E FRET signal when it is in an open state ( Fig. 1A).…”

“…In the absence of accessory proteins, UvrD, Rep, and PcrA must assemble to form at least a dimer to activate helicase activity (11,(16)(17)(18)(19)(20). Structural and solution studies show that the 2B subdomains of UvrD (22)(23)(24), Rep (25,26), and PcrA (27)(28)(29)(30) are flexible and can rotate 130 to 160°. The 2B subdomain of the Rep monomer is autoinhibitory, as its removal activates Rep monomer helicase activity (16,31,32), demonstrating that a Rep monomer possesses all that is needed for both translocase and helicase activities.…”

“…Cross-linking of the 2B subdomain of Rep into a closed configuration also activates Rep monomer helicase activity (29). Finally, upon formation of a UvrD dimer the 2B subdomain of the lead UvrD subunit is shifted to a more closed state (24).…”